Approximately 1 million total hip replacement (total hip arthroplasty) operations are carried out world-wide annually, with more than 120,000 of these undertaken in the USA, and about 35,000 in England alone (NIH Consensus Statement, 1994; NHS Review 1996). This is likely to increase to approximately 3 million worldwide per annum within the next decade. Hip replacements are very often performed in elderly patients and, amongst this group, loosening of one or both components of the prosthesis, resulting in severe mobility restriction, occurs within 15 years in about a third of patients. Where prosthetic loosening occurs, patients' experience increased pain and walking difficulty and have a higher risk of dislocations and pathological fractures. Within 10 years, approximately 10% of all patients require revision surgery, which has a high rate of complications and failure (Hellman et al, 1999).
The most common cause of implant failure is aseptic loosening as a result of particulate-induced osteolysis. Wear particles, such as particles of polyethylene, polymethylmethacrylate, titanium, cobalt chrome or ceramic debris, depending on the type of prosthesis, stimulate an inflammatory response termed periprosthetic osteolysis (Goldring et al, 1986). The phagocytosis of wear particles by macrophages activates them, leading to secretion of the inflammatory cytokines IL-1, TNF-α, and IL-6. The resulting chronic inflammatory response eventually produces a pseudomembrane of granulomatous ‘interface tissue’ including activated macrophages, fibroblasts, giant cells and osteoclasts, similar to the pannus characteristic of arthritic joints. The end result of this complex inflammatory and proliferative foreign body response is osteoclast-mediated resorption of bone, leading to loosening of one or both components of the prosthetic implant. Prostheses for total hip arthroplasty consist of two components. An artificial socket, or acetabular component, is located in a prepared cavity in the acetabulum of the pelvis. This articulates with a femoral component comprising a ball attached to a process, which is introduced into a prepared cavity in the medulla of the femur. Many variations of both components exist, and they may be retained with or without cements.
Aseptic loosening eventually leads to an unacceptable degree of pain, immobility or walking difficulties and instability, with a higher risk of dislocations and pathological fractures. In some patients revision surgery may be undertaken to remove the inflammatory tissue and replace the prosthesis. However, revision surgery is very expensive and has a high morbidity and mortality rate, especially in elderly patients (who are in the majority). In patients with cardiac insufficiency revision surgery often has major complications such as myocardial failure or coronary artery disease (Strehle et al, 2000). Many patients are not eligible for revision surgery because the risk of mortality is considered to be too high. There is no alternative treatment for such patients, who are then wheelchair-bound. The clinical need for a less traumatic alternative to revision surgery for treatment of loosened prostheses is therefore clear. At present experimental approaches to this problem are preventative rather than therapeutic. One such preventative approach to controlling aseptic loosening involves the use of bisphosphonate compounds, especially alendronate, as either a systemic medication or as a component of a cement used to fix such prostheses (U.S. Pat. No. 5,972,913, WO 96/39107, Shanbhag et al, 1997, Leung et al, 1999). However, although bisphosphonates are known to produce an increase in skeletal bone density, they have not been shown to have a significant effect in treating rheumatoid arthritis, which shares many similar pathological features with periprosthetic osteolysis, nor on periprosthetic osteolysis itself (Ralston et al, 1989; Eggelmeijer et al, 1996; Ulrich-Vinther, 2002). It thus remains to be seen whether bisphosphonates have a useful role to play in the prevention of aseptic loosening.
In an attempt to prevent osteoclast-mediated periprosthetic bone resorption directly, an alternative preventative approach involves gene therapy (reviewed in Wooley and Schwarz, 2004), using an osteoclast inhibitory protein, osteoprotegerin, delivered by means of adeno-associated virus vector has been described (Ulrich-Vinther, 2002). Osteoprotegerin is a competitive inhibitor of an osteoclast differentiation factor, receptor activator of nuclear factor κB ligand (RANKL), which binds to a receptor expressed on the surface of macrophage-derived osteoclast precursor cells, known as receptor activator of nuclear factor κB (RANK). RANKL is secreted by osteoblasts, stromal cells and activated T cells at an early stage of the inflammatory response initiated by macrophage phagocytosis of wear particles (Teitelbaum, 2000). Binding of RANKL to RANK leads to activation of osteoclast precursor cells, differentiation, and stimulation of bone resorption. Binding of RANK by osteoprotegerin fails to activate the osteoclast precursor cells with the result that osteoprotegerin competitively inhibits RANKL.
Ulrich-Vinther et al used a recombinant adeno-associated virus (rAAV) vector to express osteoprotegerin and inhibit titanium particle-induced resorption in a mouse calvarial resorption model. Titanium particles were implanted on the calvaria (bones of the vault of the skull) and the vector administered by intramuscular injection into the quadriceps. The inhibitory effect of the osteoprotegerin was therefore systemic, with detectable increases in serum levels, and this appeared to be successful in inhibiting the experimental titanium-induced osteoclastogenesis and bone resorption seen in the untreated controls. Although interesting, it remains to be seen whether this model will form the basis of a viable preventative for clinical periprosthetic osteolysis. Even if effective, it is unclear what long-term systemic effects prolonged elevations in serum osteoprotegerin levels might have. For example, such a strategy would need to demonstrate a lack of deleterious effects on normal osteoclast function in bone remodelling.
There remains a need for effective treatments for the common and debilitating condition of periprosthetic osteolysis and its resultant aseptic loosening.
One approach to preferentially killing pathological cells, most widely used for treating cancer, is to introduce a gene into the target cells that encodes an enzyme capable of converting a prodrug of relatively low toxicity into a potent cytotoxic drug. Systemic administration of the prodrug is then tolerated since it is only converted into the toxic derivative locally, for example in a tumour, by cells expressing the prodrug-converting enzyme. This approach is known as gene-directed enzyme prodrug therapy (GDEPT), or when the gene is delivered by means of a recombinant viral vector, virus-directed prodrug therapy (VDEPT) (McNeish et al, 1997).
An example of an enzyme/prodrug system is nitroreductase and the aziridinyl prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) (Knox et al 1988). Following the observation that the Walker rat carcinoma cell line was particularly sensitive to CB1954, it was shown that this was due to the expression of the rat nitroreductase DT diaphorase. However, since CB1954 is a poor substrate for the human form of this enzyme, human tumour cells are far less sensitive to CB1954. GDEPT was conceived as a way of introducing a suitable nitroreductase, preferably with greater activity against CB1954, in order to sensitise targeted cells. The Escherichia coli nitroreductase (EC1.6.99.7, alternatively known as the oxygen-insensitive NAD(P)H nitroreductase or dihydropteridine reductase, and often abbreviated to NTR) encoded by the NFSB gene (alternatively known as NFNB, NFSI, or DPRA) has been widely used for this purpose (Reviewed in Grove et al, 1999). The NFSB-encoded nitroreductase (NTR) is a homodimer that binds two flavin mononucleotide (FMN) cofactor molecules. Using NADH or NADPH as an electron donor, and bound FMN as a reduced intermediate, NTR reduces one or other of the two nitro-groups of CB 1954 to give either the highly toxic 4-hydroxylamine derivative or the relatively non-toxic 2-hydroxylamine. Within cells, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, probably via a further toxic metabolite, becomes very genotoxic (Knox et al, 1991). The exact nature of the lesion caused is unclear, but is unlike that caused by other agents. A particularly high rate of inter-strand cross-linking occurs and the lesions seem to be poorly repaired, with the result that CB1954 is an exceptionally affective anti-tumour agent (Friedlos et al, 1992).
The aim of GDEPT is to obtain efficient conversion of a prodrug such as CB1954 in target cells in order to kill not only NTR-expressing cells but also bystander tumour cells that may not have been successfully transfected or transduced.
Another enzyme-prodrug system used in this way is that of a cytochrome P450 as a prodrug-converting enzyme and acetaminophen as the prodrug, as described in international application WO 00/40271 (incorporated herein in its entirety). A number of cytochrome P450 enzymes, naturally expressed in the liver (for example CYP1A2, CYP 2E1 and CYP3A4) are capable of converting acetaminophen into a highly cytotoxic metabolite, N-acetylbenzoquinoneimine (NABQI). This system has been proposed for a variety of clinical applications, especially in the field of cancer therapy. Cytochrome P450 enzymes are also capable of activating several conventional cytotoxic prodrugs, for example cyclophosphamide and ifosfamide (Chen and Waxman, 2002).
A number of other enzyme-prodrug systems are widely used, including HSV thymidine kinase and ganciclovir (Moolten, 1986), cytosine deaminase and 5-fluorocytosine (Mullen et al., 1992).
Goossens et al (1999) describe a viral gene therapy approach to infect and kill isolated cultured synovial cells in vitro, and to kill pannus tissue in a monkey collagen-induced arthritis model in which inflamed joints are induced by collagen injections. Inflamed joints in such animals contain a hyperplastic tissue resulting from the chronic inflammation termed pannus.